Specific Aim 1. To identify the enzymatic sources of ROS generation and Nox expression in the urothelial and bladder tissues at different ages (young vs. middle-aged and aging) in experimental animals (mice) and humans. This aim will identify the origin of ROS and specific contribution from Nox enzymes, and then the expression of the Nox in the urothelium and smooth muscle, and the age dependence. These form the basis for the action of Nox-derived ROS in the bladder. 1. Major sources for ROS generation in urothelium and smooth muscle (NADPH oxidases; mitochondria; other oxidases/oxygenases) 2. Expression and activity of Nox2 (prototype) and other Nox isoforms (Nox1, Nox4) in urothelium and smooth muscle 3. Age-dependent changes to ROS generation and Nox activities. Specific Aim 2. To determine age-associated Nox activation in urothelium and consequent oxidative stress damage of bladder function at different ages using wild-type and Nox knockout mice. This aim will define Nox activation in the urothelium in response to the inflammatory factors encountered during aging, its consequent action on urothelial and smooth muscle function and the underlying mechanisms. 1. To identify the changes in urothelial Nox activity in response to inflammatory stimuli in young versus middle-aged and aging bladders 2. To characterize the effects of Nox activation on urothelial ATP release and smooth muscle contractions in young versus aging bladders 3. To discover the key signaling mediators and proteins in aging-dependent Nox activation in urothelial and bladder tissues. Specific Aim 3. To investigate the cellular mechanisms of redox-regulation in urothelial function using primary urothelial cells isolated from wild-type versus Nox knockout mice at different ages. This aim will explore further the cellular mechanisms of ROS/Nox regulation and their interaction with the key pathways for aging and bladder function, providing fundamental mechanisms for Nox contribution to bladder dysfunction during aging. 1. Effect of different ROS enzyme inhibitors and ROS scavengers on urothelial cell release of ATP and other sensory mediators. 2. Changes in urothelial Nox activity in response to inflammatory stimuli. Focus on the stress pathway. 3. Effects of ROS (H2O2) at different levels on urothelial ATP release and calcium release. 4. Importance of ROS for downstream intracellular signaling pathways in the regulation of urothelial transmitter release - role of intracellular Ca2+, exocytosis, chloride channels, kinases and connexins.